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Abstract

Background

Anopheles nili is a major vector of malaria in the humid savannas and forested areas of sub-Saharan
Africa. Understanding the population genetic structure and evolutionary dynamics of
this species is important for the development of an adequate and targeted malaria
control strategy in Africa. Chromosomal inversions and microsatellite markers are
commonly used for studying the population structure of malaria mosquitoes. Physical
mapping of these markers onto the chromosomes further improves the toolbox, and allows
inference on the demographic and evolutionary history of the target species.

Results

Availability of polytene chromosomes allowed us to develop a map of microsatellite
markers and to study polymorphism of chromosomal inversions. Nine microsatellite markers
were mapped to unique locations on all five chromosomal arms of An. nili using fluorescent in situ hybridization (FISH). Probes were obtained from 300-483 bp-long inserts of plasmid
clones and from 506-559 bp-long fragments amplified with primers designed using the
An. nili genome assembly generated on an Illumina platform. Two additional loci were assigned
to specific chromosome arms of An. nili based on in silico sequence similarity and chromosome synteny with Anopheles gambiae. Three microsatellites were mapped inside or in the vicinity of the polymorphic chromosomal
inversions 2Rb and 2Rc. A statistically significant departure from Hardy-Weinberg equilibrium, due to a
deficit in heterozygotes at the 2Rb inversion, and highly significant linkage disequilibrium between the two inversions,
were detected in natural An. nili populations collected from Burkina Faso.

Conclusions

Our study demonstrated that next-generation sequencing can be used to improve FISH
for microsatellite mapping in species with no reference genome sequence. Physical
mapping of microsatellite markers in An. nili showed that their cytological locations spanned the entire five-arm complement, allowing
genome-wide inferences. The knowledge about polymorphic inversions and chromosomal
locations of microsatellite markers has been useful for explaining differences in
genetic variability across loci and significant differentiation observed among natural
populations of An. nili.

Keywords:

Background

Anopheles gambiae, An. arabiensis, An. funestus, and An. nili are the major malaria vectors in sub-Saharan Africa because they are anthropophilic
and susceptible to Plasmodium falciparum [1-3]. These species belong to species complexes or groups, and members within these complexes/groups
vary significantly in their vectorial capacity. Moreover, species can be further sub-divided
into populations adapted to different environments. Some malaria control initiatives
have failed because they targeted the wrong species or population [4,5]. Understanding and targeting the heterogeneity and complexity of all major vector
species and populations is necessary for effective vector control and malaria eradication
[6].

Most studies of African malaria vectors have involved only An. gambiae, An. arabiensis, and An. funestus, while research on other important malaria vectors has critically lagged behind.
For An. nili, this is partly because molecular and cytogenetic tools for characterizing population
structure, ecological adaptation, and taxonomic status have been lacking. Anopheles nili is widely distributed and contributes substantially to malaria transmission in the
African savannah and forested areas, where it breeds in lotic streams and rivers [7,8]. Sporozoite rates in this species can reach 3%, and the annual entomological inoculation
rates can be over 100 [9]. For example, An. nili is highly anthropophagous and responsible for 10.2% of malaria transmission in the
densely populated area surrounding Yaounde, the capital of Cameroon [10]. Gaps in our knowledge of this vector represent a critical barrier to progress in
the field of vector biology. Recent findings of circulation of P. falciparum and other Plasmodium species in great apes and other primates [11-13] raise concerns about pathogen transfer between humans and primates, and highlight
the need to improve our knowledge of malaria vectors that inhabit forested areas in
Central Africa.

Multi-allelic microsatellites are informative markers for inferring the population
and taxonomic status of disease vectors and parasites [1,14-26]. Microsatellites are hyper-variable markers that tend to evolve neutrally. Eleven
polymorphic microsatellite markers have been developed for An. nili [27]. Recently, the level of genetic variability and differentiation has been explored
among nine populations of An. nili from Senegal, Ivory Coast, Burkina Faso, Nigeria, Cameroon, and The Democratic Republic
of Congo (DRC) [1]. Genetic variability was determined by assessing polymorphisms at these 11 microsatellite
markers, together with sequence variations in four genes within the ITS2, 28S rDNA
subunit D3, and mitochondrial DNA. High FST estimates based on microsatellites (FST > 0.118, P < 0.001) were observed in all comparisons between Kenge in the DRC, and all other
populations sampled from Senegal to Cameroon. Sequence variation in mtDNA genes matched
these results; however, low polymorphism in rDNA genes prevented detection of any
population substructure at this geographical scale. Both local adaptation and geographic
isolation could cause this differentiation. Geographic isolation should affect all
markers, even if they are unlinked (i.e. located in different chromosomes). However,
chromosomal locations of the microsatellite markers and, therefore, the degree of
their physical independence in the genome were unknown. Furthermore, because reduced
recombination and increased selection within or near polymorphic inversions can result
in estimates of gene flow that may differ significantly from those based on loci elsewhere
in the genome [28,29], it would also be important to know the location of microsatellite markers with respect
to polymorphic inversions in An. nili when performing population genetic analyses.

Polymorphic chromosomal inversions are usually under selection and, thus, are useful
markers for studying ecological adaptations of malaria mosquitoes [30-32]. The polymorphic inversions of chromosome 2 of An. gambiae have been associated with the arid Sahel Savanna [33-37] and with tolerance to desiccation and heat [38,39]. Moreover, frequencies of these inversions are higher indoors where the nocturnal
saturation deficit is higher than outdoors [35]. Such ecological heterogeneity has important consequences for vector control. For
example, indoor residual spraying of insecticides affected only indoor populations
of An. gambiae in the Garki malaria control project in Nigeria [40]. Our previous cytogenetic analysis demonstrated that two polymorphic inversions,
2Rb and 2Rc, are present simultaneously in an An. nili mosquito. However, they display very different patterns of polymorphism. Frequencies
of inverted and standard 2Rb variants were almost equal (with a deficiency of heterozygotes) in Burkina Faso, whereas
only the standard arrangement was found in Cameroon. In contrast, inversion 2Rc occurred at higher frequency (without a deficiency of heterozygotes) in the dry savannah
of Burkina Faso (83%) and at lower frequency in the humid rainforest of Cameroon (0.6%)
[32]. Moreover, inversion 2Rc was found in the mountainous area (Magba), but not in the forested area (Mbebe) of
Cameroon. These observations suggest the involvement of inversions in local adaptation
(2Rb) or in an ecogeographic adaptive cline from dry to more humid environments (2Rc). Because An. nili is a forest-savannah transition species, polymorphic inversions could provide genetic
plasticity that allows this species to expand its range from dry savannah to deforested
areas of Central Africa, where most of the human population is present. The relationship
between these two inversions has not been studied. For example, it would be useful
to know if inversions 2Rb and 2Rc are in linkage disequilibrium (LD) in natural populations of An. nili.

In this study, we mapped nine microsatellite markers to polytene chromosomes of An. nili using fluorescent in situ hybridization (FISH). Plasmid clones of the An. nili microsatellites and/or ad hoc DNA fragments amplified from a low coverage assembly of the An. nili genome were used as probes. The microsatellites hybridized to unique locations on
all chromosomes both inside and outside polymorphic inversions. We further demonstrated
highly significant linkage disequilibrium between inversions 2Rb and 2Rc. This knowledge about polymorphic inversions and chromosomal locations of microsatellite
loci helped us to better understand genetic variations and differentiation in natural
populations of An. nili.

Results

Experimental approaches to microsatellite mapping

In the current study, we used three experimental approaches to map microsatellite
markers to the polytene chromosomes from ovarian nurse cells of wild female An. nili specimens collected in Burkina Faso. In the first approach, microsatellites were amplified
from genomic DNA using specific primers, which were previously developed [27]. All microsatellites were successfully amplified from the genomic DNA. However, because
of the small size of the products (approximately 90-230 bp), a majority of the probes
failed to hybridize to chromosomes. Only one microsatellite, 1F43, was mapped by this
method. In the second approach, inserts containing microsatellites previously cloned
in the pUC18 plasmid [27] were amplified using M13 forward and reverse primers. The insert sizes in this case
ranged from 300 to 483 bp. Most of the microsatellites, except F41, B115, 2C157, and
A154 were successfully labeled and hybridized to polytene chromosomes. Marker 1F43
was also mapped by the second approach to the same chromosomal region as in the first
approach. In the third approach, we used a recently obtained genomic sequence assembly
of An. nili to identify the microsatellite loci via BLASTN search and to design primers for PCR.
These primers allowed the amplification of 506-559 bp-long PCR products containing
the microsatellites that could not be hybridized previously. The An. nili genome was sequenced by Illumina 72 bp paired-end method using genomic DNA isolated
from two individual larvae collected in Dinderesso, Burkina Faso. The assembly consisted
of 51,048 contigs with a total length of 98,320,874 bp. The average contig length
was 1,926 bp and the maximum contig length was 30,512 bp. Primers were designed for
microsatellites B115, 2C157, and A154 (accession numbers: JF742787, JF742788, JF742789)
based on sequences identified by BLASTN (Table 1). We successfully mapped microsatellites B115 and 2C157 to polytene chromosomes using
this approach. However, microsatellite A154 failed to hybridize to chromosomes despite
several attempts. The BLASTN search yielded multiple hits for microsatellite locus
F41 in the An. nili genome because of widespread occurrence of the (CT)11TT(CT)8 repeats. The BLASTN search of the flanking regions did not yield any significant
hits in the An. nili genome.

Table 1. Primers designed for the microsatellite loci using the An. nili genome sequence.

Locations of microsatellite markers on the chromosomal map of Anopheles nili

The An. nili chromosomal complement in ovarian nurse cells consists of five chromosomal arms: X,
2R, 2L, 3R and 3L. All nine microsatellites were mapped to unique locations on all
autosomes and the X chromosome using FISH (Figure 1). We assigned these microsatellites to the precise positions on the recently developed
polytene chromosome map of An. nili (Figure 2, Table 2). Two microsatellites hybridized to the X chromosome in subdivisions 2A and 3A; three
microsatellites localized to the 2R arm in subdivisions 15C, 17AB and 18A; two microsatellites
were mapped to the 3L arm in regions 38B and 44A; and arms 2L and 3R each hybridized
with only one microsatellite marker in sections 20C and 31C, respectively. Only one
microsatellite, 2C157, was mapped inside the previously described polymorphic inversion
2Rc. Microsatellite 1A27 localized to subdivision 15C located between inversions 2Rb and 2Rc. Microsatellite 1F43 was mapped to subdivision 18A located next to the proximal breakpoint
of inversion 2Rc.

Figure 1.FISH of microsatellites performed on the An. nili chromosomes. Hybridizations of microsatellite markers 1G13 (A), 2Ateta and 1F43 (B), and B115
(C) with polytene chromosomes are shown. Chromosomes were counterstained with the
fluorophore YOYO-1 and hybridized with fluorescently labeled probes Cy5 (blue) and
Cy3 (red). The top panel shows fluorescent images of chromosomes after FISH. The bottom
panel shows inverted grayscale chromosome images with color labels and distinct banding
patterns.

Figure 2.Physical chromosome map of the An. nili microsatellites. Chromosomal locations of nine microsatellite markers on polytene chromosomes are
shown by arrows. Two polymorphic chromosomal inversions are indicated by brackets.

In this study, we identified sequences in the An. gambiae genome that are homologous to six microsatellite loci of An. nili (Table 2). The remaining five loci did not have significantly similar sequences in the An. gambiae genome. Markers A14, 2C157, 2Ateta, and B115, which we mapped in An. nili by FISH, were placed to specific regions of homologous chromosome arms in An. gambiae by BLASTN. The BLASTN results confirmed arm homologies between the two species that
we determined in our previous study [32]. In addition, we mapped in silico microsatellites A154 and F41, which were not previously mapped by FISH. We used the
Illumina-based genome sequence assembly of An. nili for A154 and the clone sequence for F41 to perform BLASTN against the An. gambiae genome. According to the established arm homology, we assigned microsatellites A154
and F41 to 3R and 2R chromosome arms of An. nili, respectively (Table 2).

Inversion polymorphism in Anopheles nili

To test if inversions 2Rb and 2Rc are in LD, we karyotyped 44 An. nili females collected in Dinderesso, Burkina Faso. Inversion frequencies were calculated
jointly for these individuals and for 56 previously karyotyped females from the same
village [32]. We found a highly significant LD between the two inversions (P = 0.00054), i.e.,
these inversions occur together much more often than expected. Frequencies of inverted
and standard 2Rb variants were almost equal (0.51 and 0.49 for the standard and inverted arrangement,
respectively). However, a highly significant departure from Hardy-Weinberg proportions
due to a deficit in heterozygotes (e.g., positive FIS value) was observed at this locus (FIS = +0.603, P < 0.0001 single test level). Inversion 2Rc occurred at high frequency in the sample (0.825), with no significant deviation from
Hardy-Weinberg equilibrium (HWE) (P = 0.49) (Table 3).

Discussion

Availability of readable polytene chromosomes in An. nili allowed us to develop a map of microsatellite markers and to study polymorphism of
chromosomal inversions. Among the three experimental approaches used to map microsatellite
markers to chromosomes, using cloned inserts and genome sequence assembly of An. nili to amplify and hybridize microsatellites was more successful than using microsatellite
fragments amplified with primers for population genetics studies [1,27] (Figure 1). Larger DNA fragments were more suitable for effective labeling by the random primer
method than smaller fragments obtained with primers for population genetic studies
[27]. In addition to these experimental approaches, we conducted BLASTN searches of the
An. nili genome fragments with microsatellites (both Illumina generated and cloned) against
the An. gambiae genome to assign microsatellite loci to chromosome arms according to the synteny between
An. nili and An. gambiae [32] (Table 2). Although, X, 2R, and 3R are homologous between the two species, the 2L arm of An. gambiae corresponds to the 3L arm of An. nili, and the 3L arm of An. gambiae corresponds to the 2L arm of An. nili, indicating the presence of a whole-arm translocation. Because of the high number of
inversions fixed between the two species, the genome of An. gambiae cannot be used as a reference for precise positioning of microsatellites on the An. nili chromosomes. In our previous study, we calculated the minimum number of fixed inversions
among An. nili, An. gambiae, and An. stephensi and concluded that An. nili is, at least, as diverged from An. gambiae as An. stephensi [32]. In addition to the fixed inversion differences, An. nili has a distinct pattern of polymorphic inversions. Therefore, the chromosomal positions
of homologous loci with respect to polymorphic inversions will be different in the
two species.

The developed microsatellite map (Figure 2) improved our understanding of the population genetic structure of An. nili. A recent study using 11 microsatellite markers demonstrated significant genetic differentiation
of the An. nili population of Kenge in the DRC as compared to the An. nili populations in Central and West Africa [1]. Both local adaptation and geographic isolation could cause this differentiation.
Extensive allele sharing between populations and homogeneity across loci suggested
that enhanced genetic drift rather than selection was responsible for the observed
pattern. Although it is unlikely that all loci would be within or close to the same
inversion, chromosomal mapping of the markers was needed to determine the degree of
their independence. Our study demonstrated that the microsatellite locations are not
limited to one or a few specific regions in the genome but spanned the entire five-arm
complement (Figure 2). Because most of these markers are physically unlinked, we conclude that enhanced
genetic drift, rather than selection was responsible for reduced variability and increased
differentiation of the Kenge, DRC population (see also Additional file 1). These data strongly suggest the role of the equatorial forest block as a barrier
to gene flow between the south-African and north-African populations of An. nili.

Additional file 1.Re-analysing genetic differentiation between Anopheles nili populations from West and Central Africa. The file contains the genotypic data re-analyzed according to microsatellite loci
cytological location. Locus-specific FST values are shown in Table S1, together with FST estimates across each chromosomal arm and overall. Locus-specific jackknifed mean
FST estimates (+/- standard deviation) between An. nili populations from West and Central Africa are shown in Figure S1.

Among the mapped microsatellite loci, 1A27 and A14 were found to be in particularly
strong and significant departure from HWE due to a deficiency of heterozygotes in
West Africa (Burkina Faso and Senegal) but not in Central Africa (Cameroon) [1]. We also detected a highly statistically significant departure from HWE due to a
deficit in heterozygotes (FIS = +0.603, P < 0.0001 single test level, Table 3) at inversion 2Rb in the village of Dinderesso in Burkina Faso among 100 karyotyped females. It is possible
that the 2Rb inversion plays a role in local adaptation and subdivides An. nili into populations with limited gene flow. This process or the presence of null alleles
could cause heterozygote deficiency at microsatellite loci. In contrast, inversion
2Rc demonstrated no significant deviation from HWE (Table 3). However, we found a highly significant LD between the two inversions (P = 0.00054).
Microsatellite 1A27 is located between 2Rb and 2Rc and it could be affected by the LD and reduced recombination in the vicinity of chromosomal
breakpoints (Figure 2). Future studies should determine whether this LD is caused by physical linkage or
selection. 2C157 is the only microsatellite located inside an inversion; and it does
not demonstrate deficiency of heterozygotes. This locus is in the middle of inversion
2Rc where recombination could be close to normal. Moreover, significant departure from
HWE due to a deficiency of heterozygotes was demonstrated for inversion 2Rb but not for 2Rc. Marker A14 is located on the X chromosome, which lacks polymorphic inversions, suggesting
that genetic differentiation is not limited to the inversions (see Additional file
1 for locus-specific FST estimates). Microsatellites in Hardy-Weinberg disequilibrium could also be associated
with genes responsible for epidemiologically important ecological adaptations. Indeed,
the microsatellite motif of A14 is located 259 bp upstream from the start codon of
an open reading frame in the An. nili genome, and the sequence homologous to the A14 clone is found in the 5'UTR and the
first exon of the An. gambiae gene AGAP000275. According to gene ontology annotation, the protein encoded by this
gene has oxidoreductase activity. The transcript of AGAP000275 has demonstrated significant
differential expression in a variety of mosquito tissues and life stages. Significant
differences have been shown between: different stages of embryonic development, between
embryonic serosa and embryo [41], between blood-fed and non-blood-fed females, between fat body and ovaries, between
males and females, between adults and larva [42], between hemolymph and carcass [43], between West and East African strains of S form gravid females [44], between larval anterior midgut and hindgut [45], between larval salivary gland and whole organism [46]. Significant 1.2-fold increase in the transcription level of AGAP000275 has also
been found between females 6 hours and 24 hours after mating [47]. Altogether, these data suggest strong selection acting on AGAP000275 in An. gambiae that might translate into non-neutral polymorphism distribution at locus A14 in An. nili. Sequences homologous to other An. nili microsatellite loci with significant BLASTN hits in the An. gambiae genome were found outside genes, except microsatellite B115, which was located within
the second intron of gene AGAP004824.

Our recent mapping of 12 microsatellites to An. stephensi chromosomes has demonstrated that the chromosomal position of microsatellites may
affect estimates of population genetic parameters [48]. In a similar study of An. funestus, 16 microsatellites were physically mapped to polytene chromosomes, and the location
of microsatellites based on the inversions were determined [49]. Interestingly, microsatellites located between inversions 3Ra and 3Rb in An. funestus were found in LD with these inversions in Burkina Faso [50] but not in Cameroon [30], reflecting different evolutionary outcomes in different eco-geographic regions.
Altogether, these studies point to the importance of physical mapping of molecular
markers exposed to contrasted evolutionary dynamics for unravelling the demographic
and evolutionary history of malaria vectors. This paper provides the necessary toolbox
for such endeavour to be pursued in An. nili.

Conclusions

Our study demonstrates that the chromosomal position of microsatellites is informative
for interpretation of population genetics data and highlights the importance of developing
physical maps for nonmodel organisms. Next-generation sequencing can be used for designing
microsatellite primers to obtain longer microsatellite-containing probes and improve
FISH mapping. An Illumina-based genome sequence assembly can also be used for identifying
homologous loci in the reference genomes and assigning microsatellite markers to chromosomal
arms in a species of interest based on synteny. The integrated chromosomal map of
microsatellites and inversions will allow for more complete characterization of An. nili in future population genetics studies. It will be possible to test for a LD among
and between inversions and microsatellites, genetic differentiation at microsatellite
loci located inside and outside inversions, and genetic differentiation according
to the distance from inversion breakpoints. In addition, the new genetic map could
be used for designing quantitative trait loci mapping studies for this species.

Methods

Wild mosquito collection, preservation, and species identification

Anopheles nili adult females were collected by pyrethrum spraying and bednet traps in the village
of Dinderesso (11°14'N; 4°23'W) in Burkina Faso. Anopheles nili larvae were collected in a river in Dinderesso, Burkina Faso. Specimens were identified
in the field as members of the An. nili group by using morphological identification keys [51-53] and were further characterized by molecular assays as An. nili s.s. [54]. Females were dissected under a microscope, and their ovaries at the appropriate
stage were preserved in Carnoy's fixative solution (3 parts of ethanol: 1 part of
glacial acetic acid by volume). Ovaries were kept at room temperature overnight before
being stored at -20°C. Larvae were preserved in Carnoy's fixative solution and stored
at -20°C.

Genome sequencing and BLASTN

The genome assembly for An. nili was obtained by sequencing of genomic DNA isolated from two larvae collected in Dinderesso,
Burkina Faso. Genomic DNA was isolated using the Qiagen DNeasy Blood and Tissue Kit
(Qiagen Science, Germantown, MD, USA). The library preparation and sequencing was
performed on the Illumina Genome Analyzer IIx, using 72 bp paired-end processing at
Ambry Genetics Corp. (Aliso Viejo, CA, USA). Samples were prepared using the Illumina
protocol outlined in "Preparing Samples for Sequencing Genomic DNA" (Part # 11251892
Rev. A 2007). Briefly, DNA fragment ends were repaired and phosphorylated using Klenow,
T4 DNA Polymerase and T4 Polynucleotide Kinase. Next, an 'A' base was added to the
3' end of the blunted fragments, followed by ligation of Illumina paired-end adaptor
via T-A mediated ligation. The ligated products were size selected by gel purification
and then PCR amplified using Illumina Paired-End primers. The library size and concentration
were determined using an Agilent Bioanalyzer. The library was seeded onto the flowcell
at 8 pM, yielding approximately 275 K clusters per tile, and it was sequenced using
73 cycles of chemistry and imaging (73 cycles) for read 1 and read 2. Initial data
processing, including extraction of cluster intensities and base calling, was done
using RTA 1.6.47 (SCS version 2.6.26). Sequence quality filtering scripts were executed
in the Illumina CASAVA software (ver 1.6.0, Illumina, Hayward, CA). Quality metric
data included the approximate proportion of sequences with 1, 2, 3 or 4 errors, IVC
plots, and visualizations of cluster intensity over the duration of the sequencing
run. The BLASTN algorithm was used to identify homologous sequences in the An. gambiae genome, which is available at VectorBase [55]. The BLASTN algorithm was also used to find larger genomic fragments with microsatellite
loci in the An. nili genome using a server and the Geneious 5.1.5 software http://www.geneious.comwebcite, a bioinformatics desktop software package produced by Biomatters Ltd http://www.biomatters.comwebcite.

Probe preparation

Three approaches were utilized for the microsatellite probe preparation. First, microsatellites
were directly amplified from the An. nili genomic DNA using previously designed primers [27]. Approximately 90-230 bp-long fragments were amplified. Second, plasmid clones with
microsatellites were used as templates for insert amplification. In this case, 300-483
bp-long fragments were amplified from the pUC18 plasmid DNA using standard M13 forward
and reverse primers (Fermentas, Inc., Glen Burnie, MD, USA). Third, primers were designed
for three microsatellites using the Primer3 program [56] based on sequences identified by BLASTN in the genome assembly of An. nili (accession numbers: JF742787, JF742788, JF742789). The size of these fragments was
about 506-559 bp. PCR conditions were as follows: 94°C for 5 min; 45 cycles of 94°C
for 45 s, 50°C for 45 s and 72°C for 30 s; and 72°C for 5 min. DNA was purified using
the GE healthcare illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare
UK Ltd, Buckinghamshire, UK). Probes were labeled using Cy3-AP3-dUTP or Cy5-AP3-dUTP
(GE Healthcare UK Ltd., Buckinghmashire, UK) fluorophores by a Random Primer DNA Labelling
System (Invitrogen Corporation, Carlsbad, CA, USA).

Chromosome preparation and FISH

To obtain chromosomal slides, follicles of ovaries were separated in 50% propionic
acid. Then a cover slip was used to squash the follicles. The quality of slides and
the banding pattern of polytene chromosomes were analyzed using an Olympus CX-41 phase
contrast microscope (Olympus America Inc., Melville, NY, USA). Slides then were dipped
into liquid nitrogen, cover slips were removed, and slides were dehydrated in 50%,
70%, 90% and 100% ethanol. Slides were air dried and used for further experiments.
Labelled probes were hybridized at 42°C to An. nili polytene chromosome slides overnight. Then, slides were washed in 0.2 X SSC (Saline
Sodium citrate, 0.03 M sodium chloride and 0.03 M sodium citrate) at 42°C and room
temperature. Chromosomes were stained using YOYO-1 (Invitrogen Corporation, Carlsbad,
CA, USA), and slides were mounted in 1,4-diazabicyclo[2.2.2]octane (DABCO) antifade
solution. A Zeiss LSM 510 Laser Scanning Microscope (Carl Zeiss MicroImaging, Inc.,
Thornwood, NY, USA) was used to detect fluorescent signals. Microscopic images were
taken from the signal, and the locations of signals were determined using a standard
cytogenetic photo map of An. nili [32].

Image processing

Confocal images were processed using ImageJ and Adobe Photoshop software as described
elsewhere [57]. Briefly, color channels were split from the initial RGB image into separate images.
Each channel image was converted into the monochrome image by using a 'Channel mixer'
and then inverted. The inverted monochrome image was adjusted by using a 'Curves'
tool until the background is removed and each chromosome of the spread becomes fuzzy-edged.
The reduction of noise was achieved by blurring of each pixel with the Gaussian blur
filter tool. The quality of the image was improved by additional application of the
'Curves' and/or subtraction of the 'Relative white'. Finally, green channel image
with chromosomes was merged with monochrome image FISH signals. Processing yielded
contrasted, inverted, grayscale images with color labels, which are more suitable
for mapping.

Population genetics analyses

Homozygous and heterozygous inversions were scored using the chromosomal map published
earlier [32]. Alternative chromosomal arrangements were considered as different alleles of the
same locus, and conformance to Hardy-Weinberg equilibrium was tested with Fisher's
exact tests available in GENEPOP V4.0 [58]. A FIS value was computed as in [59]. LD between the inversions 2Rb and 2Rc was assessed using the log likelihood ratio statistic (G-test) available in GENEPOP V4.0 [58].

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

IVS designed research; AP, IVS, and MVS performed karyotyping and physical mapping;
CAN and CN conducted field work and mosquito identification; FS, MVS, and IVS analyzed
data; MW prepared plasmid DNA; AP and IVS prepared genomic DNA for sequencing; IVS
performed BLASTN searches using VectorBase and a Geneious software; AP and IVS wrote
the paper, which was critically revised by CAN, FS, and MVS. All authors read and
approved the final manuscript.

Acknowledgements

We are grateful to Z. Tu for hosting the genome sequence of An. nili on his server and conducting BLASTN. We thank S. Demin for explaining the image processing
procedure. This work was supported by the National Institute of Allergy and Infectious
Diseases, National Institutes of Health (grant R21AI079350 to IVS).